Problem Addressed
Blood loss is a serious complication of major surgeries such as open heart surgery and other complicated procedures. Cardiac surgery patients account for a significant proportion of transfused donor blood. Blood transfusion carries risks of disease transmission and adverse reactions. In addition, donor blood is expensive and demands often exceed supply. Pharmacological methods for reducing blood loss and the resultant need for transfusion have been described (reviewed by Scott et al., Ann. Thorac. Surg. 50: 843-851,1990).
Protein Serine Protease Inhibitor
Aprotinin, a bovine serine protease inhibitor of the Kunitz family is the active substance in the medicament TRASYLOL®. Aprotinin (TRASYLOL®) has been reported as being effective in reducing perioperative blood loss (Royston et al. (1987) Lancet 2, 1289-1291; Dietrich et al., Thorac. Cardiovasc. Surg. 37: 92-98, 1989; Fraedrich et al., Thorac. Cardiovasc, Surg. 37: 89-91, 1989); W. van Oeveren et al. (1987), Ann Thorac. Surg. 44, pp 640-645; Bistrup et al., (1988) Lancet I, 366-367), but adverse effects, including hypotension and flushing (Bohrer et al., Anesthesia 45: 853-854, 1990) and allergic reactions (Dietrich et al., Supra) have been reported. Use of aprotinin in patients previously exposed to it is not recommended (Dietrich et al., Supra). TRASYLOL® has also been used for the treatment of hyperfibrinolytic hemorrhages and traumatic hemorrhagic shock.
Aprotinin is known to inhibit several serine proteases including trypsin, chymotrypsin, plasmin and kallikrein, and is used therapeutically in the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction (Trapnell et al., (1974) Brit J. Surg. 61: 177; J. McMichan et al., (1982) Circulatory Shock 9: 107; Auer et al, (1979) Acta Neurochir. 49: 207; Sher (1977) Am J. Obstet. Gynecol. 129: 164; Schneider (1976), Artzneim.-Firsch. 26: 1606). It is generally thought that Trasylol® reduces blood loss in vivo through inhibition of kallikrein and plasmin. It has been found that aprotinin (3-58, Arg15, Ala17, Ser42) exhibits improved plasma kallikrein inhibitory potency as compared to native aprotinin itself (WO 89/10374).
Problems with Aprotinin
Because aprotinin is of bovine origin, there is a finite risk of inducing anaphylaxis in human patients upon re-exposure to the drug. Thus, a human functional equivalent to aprotinin, by virtue of a lower risk of anaphylaxis, would be most useful and desirable to have.
Aprotinin is also nephrotoxic in rodents and dogs when administered repeatedly at high dose (Bayer, TRASYLOL®, Inhibitor of proteinase; Glasser et al., in “Verhandlungen der Deutschen Gesellschaft fur Innere Medizin, 78. Kongress”,Bergmann, Munchen, 1972 pp. 1612-1614). One hypothesis ascribes this effect to the accumulation of aprotinin in the negatively charged proximal tubules of the kidney,due to its high net positive charge (WO 93/14120).
Accordingly, an object of the present invention is to identify human proteins with functional activity similar to aprotinin. It was also an object of the instant invention to identify human proteins, that would be less charged, yet exhibit the same, highly similar, or improved protease specificities as found for aprotinin, especially with respect to the potency of plasmin and kallikrein inhibition. Such inhibitors could then be used repeatedly as medicaments in human patients with reduced risk of adverse immune response and reduced nephrotoxicity.